Osmoadaptation Strategy of the Most Halophilic Fungus,Wallemia ichthyophaga,Growing Optimally at Salinities above 15% NaCl

Wallemia ichthyophaga is a fungus from the ancient basidiomycetous genus Wallemia (Wallemiales, Wallemiomycetes) that grows only at salinities between 10% (wt/vol) NaCl and saturated NaCl solution. This obligate halophily is unique among fungi. The main goal of this study was to determine the optimal salinity range for growth of the halophilic W. ichthyophaga and to unravel its osmoadaptation strategy. Our results showed that growth on solid growth media was extremely slow and resulted in small colonies. On the other hand, in the liquid batch cultures, the specific growth rates of W. ichthyophaga were higher, and the biomass production increased with increasing salinities. The optimum salinity range for growth of W. ichthyophaga was between 15 and 20% (wt/vol) NaCl. At 10% NaCl, the biomass production and the growth rate were by far the lowest among all tested salinities. Furthermore, the cell wall content in the dry biomass was extremely high at salinities above 10%. Our results also showed that glycerol was the major osmotically regulated solute, since its accumulation increased with salinity and was diminished by hypo-osmotic shock. Besides glycerol, smaller amounts of arabitol and trace amounts of mannitol were also detected. In addition, W. ichthyophaga maintained relatively small intracellular amounts of potassium and sodium at constant salinities, but during hyperosmotic shock, the amounts of both cations increased significantly. Given our results and the recent availability of the genome sequence, W. ichthyophaga should become well established as a novel model organism for studies of halophily in eukaryotes.     

The Halophilic Fungus Hortaea werneckii and the Halotolerant Fungus Aureobasidium pullulans Maintain Low Intracellular Cation Concentrations in Hypersaline Environments

Hortaea werneckii and Aureobasidium pullulans, black yeast-like fungi isolated from hypersaline waters of salterns as their natural ecological niche, have been previously defined as halophilic and halotolerant microorganisms, respectively. In the present study we assessed their growth and determined the intracellular cation concentrations of salt-adapted and non-salt-adapted cells of both species at a wide range of salinities (0 to 25% NaCl and 0 to 20% NaCl, respectively). Although 5% NaCl improved the growth of H. werneckii, even the minimal addition of NaCl to the growth medium slowed down the growth rate of A. pullulans, confirming their halophilic and halotolerant nature. Salt-adapted cells of H. werneckii and A. pullulans kept very low amounts of internal Na⁺ even when grown at high NaCl concentrations and can be thus considered Na⁺ excluders, suggesting the existence of efficient mechanisms for the regulation of ion fluxes. Based on our results, we can conclude that these organisms do not use K⁺ or Na⁺ for osmoregulation. Comparison of cation fluctuations after a hyperosmotic shock, to which nonadapted cells of both species were exposed, demonstrated better ionic homeostasis regulation of H. werneckii compared to A. pullulans. We observed small fluctuations of cation concentrations after a hyperosmotic shock in nonadapted A. pullulans similar to those in salt-adapted H.werneckii, which additionally confirmed better regulation of ionic homeostasis in the latter. These features can be expected from organisms adapted to survival within a wide range of salinities and to occasional exposure to extremely high NaCl concentrations, both characteristic for their natural environment.     

Branchial Na,K-ATPase and osmoregulation in the purple shore crab,Hemigrapsus nudus (Dana)

The purple shore crab, Hemigrapsus nudus, controls its hemolymph osmolality over a wide range of external salinities: it is a strong hyperosmoregulator in 25%, 50% and 75% sea water (SW) and is isosmotic in 100% SW. The role of branchial sodium + potassium-activated, magnesium-requiring adenosine triphosphatase (NA, K-ATPase) in osmoregulation was investigated by assaying enzyme-specific activity (SEA) in gills from crabs acclimated for 14 d in the four sea water media. Assay conditions were characterized for optimal ESA with crude homogenates of gills; ion and cofactor requirements were found to be similar to those of other crustacean Na, K-ATPases. Branchial ESA was highest in crabs acclimated for 2 weeks in 50% SW and was significantly correlated with the osmotic gradient across the body wall in 50%, 75% and 100% SW. Gills 6, 7 and 8 had the highest ESA in all media and possessed approximately 70% of the total branchial Na, K-ATPase activity, but all gills showed significant, approximately twofold increases of ESA in 50% SW compared with values in 100% SW. The time courses of increased branchial Na, K-ATPase ESA and decreased hemolymph osmotic pressure in crabs transferred from 100% SW to 50% SW are consistent with both increased in vivo activity of existing enzyme in the short term and a longer-term synthesis of new enzyme by the gills which is measured by our in vitro assay.     

Oil-bioremediation potential of two hydrocarbonoclastic, diazotrophic Marinobacter strains from hypersaline areas along the Arabian Gulf coasts

Two halophilic, hydrocarbonoclastics bacteria, Marinobacter sedimentarum and M. flavimaris, with diazotrophic potential occured in hypersaline waters and soils in southern and northern coasts of Kuwait. Their numbers were in the magnitude of 10³ colony forming units g^?1. The ambient salinity in the hypersaline environments was between 3.2 and 3.5 M NaCl. The partial 16S rRNA gene sequences of the two strains showed, respectively, 99 and 100 % similarities to the sequences in the GenBank. The two strains failed to grow in the absence of NaCl, exhibited best growth and hydrocarbon biodegradation in the presence of 1 to 1.5 M NaCl, and still grew and maintained their hydrocarbonoclastic activity at salinities up to 5 M NaCl. Both species utilized Tween 80, a wide range of individual aliphatic hydrocarbons (C₉–C₄₀) and the aromatics benzene, biphenyl, phenanthrene, anthracene and naphthalene as sole sources of carbon and energy. Experimental evidence was provided for their nitrogen-fixation potential. The two halophilic Marinobacter strains successfully mineralized crude oil in nutrient media as well as in hypersaline soil and water microcosms without the use of any nitrogen fertilizers.     

Responses of Atriplex spongiosa and Suaeda monoica to Salinity

The growth and tissue water, K⁺, Na⁺, Cl⁻, proline and glycinebetaine contents of the shoots and roots of two Chenopodiaceae, Atriplex spongiosa and Suaeda monoica have been measured over a range of external NaCl salinities. Both species showed some fresh weight response to low salinity mainly due to increased succulence. S. monoica showed both a greater increase in succulence (at low salinities) and tolerance of high salinities than A. spongiosa. Both species had high affinities for Na⁺ and maintained constant but low shoot K⁺ contents with increasing salinity. These trends were more marked with S. monoica in which Na⁺ stimulated the accumulation of K⁺ in roots. An association between high leaf Na⁺ accumulation, high osmotic pressure, succulence, and a positive growth response at low salinities was noted. Proline accumulation was observed in shoot tissues with suboptimal water contents. High glycinebetaine contents were found in the shoots of both species. These correlated closely with the sap osmotic pressure and it is suggested that glycinebetaine is the major cytoplasmic osmoticum (with K⁺ salts) in these species at high salinities. Na⁺ salts may be preferentially utilized as vacuolar osmotica.     

The Comparative Osmoregulatory Ability of Two Water Beetle Genera Whose Species Span the Fresh-Hypersaline Gradient in Inland Waters (Coleoptera: Dytiscidae,Hydrophilidae)

A better knowledge of the physiological basis of salinity tolerance is essential to understanding the ecology and evolutionary history of organisms that have colonized inland saline waters. Coleoptera are amongst the most diverse macroinvertebrates in inland waters, including saline habitats; however, the osmoregulatory strategies they employ to deal with osmotic stress remain unexplored. Survival and haemolymph osmotic concentration at different salinities were examined in adults of eight aquatic beetle species which inhabit different parts of the fresh—hypersaline gradient. Studied species belong to two unrelated genera which have invaded saline waters independently from freshwater ancestors; Nebrioporus (Dytiscidae) and Enochrus (Hydrophilidae). Their osmoregulatory strategy (osmoconformity or osmoregulation) was identified and osmotic capacity (the osmotic gradient between the animal’s haemolymph and the external medium) was compared between species pairs co-habiting similar salinities in nature. We show that osmoregulatory capacity, rather than osmoconformity, has evolved independently in these different lineages. All species hyperegulated their haemolymph osmotic concentration in diluted waters; those living in fresh or low-salinity waters were unable to hyporegulate and survive in hyperosmotic media (> 340 mosmol kg^-1). In contrast, the species which inhabit the hypo-hypersaline habitats were effective hyporegulators, maintaining their haemolymph osmolality within narrow limits (ca. 300 mosmol kg^-1) across a wide range of external concentrations. The hypersaline species N. ceresyi and E. jesusarribasi tolerated conductivities up to 140 and 180 mS cm^-1, respectively, and maintained osmotic gradients over 3500 mosmol kg^-1, comparable to those of the most effective insect osmoregulators known to date. Syntopic species of both genera showed similar osmotic capacities and in general, osmotic responses correlated well with upper salinity levels occupied by individual species in nature. Therefore, osmoregulatory capacity may mediate habitat segregation amongst congeners across the salinity gradient.     

Osmoregulatory adjustments by three atherinids (Leptatherina presbyteroides; Craterocephalus mugiloides; Leptatherina wallacei) to a range of salinities

• 1.1. Patterns of osmoregulation were studied in three species of Swan river atherinids (Leptatherina presbyteroides, lower estuarine and marine; Craterocephalus mugiloides, mid estuarine; Leptatherina wallacei, upper estuarine) over a wide range of salinities.
• 2.2. The plasma Na⁺ concentration was elevated with an increase in salinity.
• 3.3. Haematocrit and body water content decreased with acclimation to higher salinity.
• 4.4. All three species of atherinids osmotically regulated over a salinity range greater than that which these fish are reported to occur in.

     

Adaptive diversity in congeneric coastal crabs: Ontogenetic patterns of osmoregulation match life-history strategies in Armases spp (Decapoda, Sesarmidae)

Ontogeny of osmoregulation and salinity tolerance were investigated throughout the larval development of two congeneric species of sesarmid crab, Armases ricordi (H. Milne Edwards) and A. roberti (H. Milne Edwards), and compared with previous observations from two further congeners, A. miersii (Rathbun) and A. angustipes (Dana). In the semiterrestrial coastal species A. ricordi, the zoeal stages were only at moderately reduced salinities (17-25.5‰) capable of hyper-osmoregulation, being osmoconformers at higher concentrations. The megalopa was the first ontogenetic stage of this species, which exhibited significant hyper-osmoregulation at further reduced salinities (≥ 5‰), as well as a moderately developed function of hypo-regulation at high concentrations (32-44‰). The riverine species A. roberti showed similar overall patterns in the ontogeny of osmoregulation, however, also some striking differences. In particular, its first zoeal stage showed already at hatching a strong capability of hyper-osmoregulation in salinities down to 5‰. Interestingly, this early expressed function became significantly weaker in the subsequent zoeal stages, where survival and capabilities of hyper-osmoregulation were observed only at salinities down to 10‰. The function of hyper-regulation in strongly dilute media re-appeared later, in the megalopa stage, which tolerated even an exposure to freshwater (0.2‰). Differential species- and stage-specific patterns of osmoregulation were compared with contrasting life styles, reproductive behaviours, and life-history strategies. In A. ricordi, the larvae are released into coastal marine waters, where salinities are high, and thus, no strong hyper-osmoregulation is needed throughout the zoeal phase. The megalopa stage of this species, by contrast, may invade brackish mangrove habitats, where osmoregulatory capabilities are required. Strong hyper-osmoregulation occurring in both the initial and final larval stages (but not in the intermediate zoeal stages) of A. roberti correspond to patterns of ontogenetic migration in this species, including hatching in freshwater, larval downstream transport, later zoeal development in estuarine waters, and final re-immigration of megalopae and juvenile crabs into limnic habitats, where the conspecific adults live. Similar developmental changes in the ecology and physiology of early life-history stages seem to occur also in A. angustipes. A. miersii differs from all other species, showing an early expression and a gradual subsequent increase of the function of hyper-osmoregulation. This ontogenetic pattern corresponds with an unusual reproductive biology of this species, which breeds in supratidal (i.e. land-locked) rock pools, where variations in salinity are high and unpredictable. Matching patterns in the ontogeny of osmoregulation and life-history strategies indicate a crucial adaptive role of osmoregulation for invasions of (by origin marine) crabs into brackish, limnic and terrestrial environments.     

Biodegradation of polycyclic aromatic hydrocarbons by a halophilic microbial consortium

In this study we investigated the phenanthrene degradation by a halophilic consortium obtained from a saline soil sample. This consortium, named Qphe, could efficiently utilize phenanthrene in a wide range of NaCl concentrations, from 1% to 17% (w/v). Since none of the purified isolates could degrade phenanthrene, serial dilutions were performed and resulted in a simple polycyclic aromatic hydrocarbon (PAH)-degrading culture named Qphe-SubIV which was shown to contain one culturable Halomonas strain and one unculturable strain belonging to the genus Marinobacter. Qphe-SubIV was shown to grow on phenanthrene at salinities as high as 15% NaCl (w/v) and similarly to Qphe, at the optimal NaCl concentration of 5% (w/v), could degrade more than 90% of the amended phenanthrene in 6 days. The comparison of the substrate range of the two consortiums showed that the simplified culture had lost the ability to degrade chrysene but still could grow on other polyaromatic substrates utilized by Qphe. Metabolite analysis by HPLC and GC–MS showed that 2-hydroxy 1-naphthoic acid and 2-naphthol were among the major metabolites accumulated in the Qphe-SubIV culture media, indicating that an initial dioxygenation step might proceed at C1 and C2 positions. By investigating the growth ability on various substrates along with the detection of catechol dioxygenase gene, it was postulated that the uncultured Marinobacter strain had the central role in phenanthrene degradation and the Halomonas strain played an auxiliary role in the culture by utilizing phenanthrene metabolites whose accumulation in the media could be toxic.     

CO(2) Fixation Rate and RuBisCO Content Increase in the Halotolerant Cyanobacterium, Aphanothece halophytica, Grown in High Salinities

The growth of the halotolerant cyanobacterium Aphanothece halophytica, previously adapted to 0.5 molar NaCl, was optimal when NaCl concentration in culture medium was in the range 0.5 to 1.0 molar. The growth was delayed at either too low or too high salinities with lag time of ca. 0.5 day in 0.25 molar NaCl and ca. 2 days in 2 molar NaCl under the experimental conditions. However, the growth rates at the logarithmic phase were similar in the culture media containing NaCl in the range 0.25 to 2.0 molar. The capacity of photosynthetic CO₂ fixation increased 3.7-fold in the cells at the logarithmic phase as NaCl concentration in the culture medium increased from 0.25 to 2.0 molar. The protein level of ribulose 1,5-bisphosphate carboxylase/oxygenase was also found to increase with increasing salinity using both an immunoblotting method and protein A-gold immunoelectron microscopy. These results indicate that high photosynthetic capacity and high ribulose 1,5-bisphosphate carboxylase/oxygenase content may entail an important role in betaine synthesis and adaptation of the A. halophytica cells to high NaCl level.     

Hydromineral regulation in the saline water corixid Trichocorixa reticulata (Hemiptera: Corixidae)

The aquatic corixid Trichocorixa reticulata (Guerin-Meneville) inhabits coastal marshes, brackish water ponds and salt ponds of high salinity, suggesting the presence of well developed mechanisms for hydromineral regulation.Groups of corixids acclimated in salinities ranging from fresh water to just above 300% sea water (100‰) were analyzed for total body water content, haemolymph ionic and osmotic levels, and haemolymph free amino acids.Results indicate an excellent ability to maintain haemolymph Na⁺, Cl^?, Mg²⁺ and K⁺ hyperosmotic to the medium at low salinities and hyposmotic at high salinities. Calcium appears to conform closely to changes in external medium, becoming hyposmotic at very high salinities (80‰).Total haemolymph osmotic pressure was well regulated, the freezing point depression varying from 0.75°C in distilled water to 1.15°C in salinities of 100‰. Total body water was maintained at approx. 75% of the total animal wet weight at all salinities tested.Free amino acids were maintained between 40–60 mM in all tests and did not appear to change with salinity.     

Molecular Characterization of the α-Subunit of Na+/K+ ATPase from the Euryhaline Barnacle Balanus improvisus Reveals Multiple Genes and Differential Expression of Alternative Splice Variants

The euryhaline bay barnacle Balanus improvisus has one of the broadest salinity tolerances of any barnacle species. It is able to complete its life cycle in salinities close to freshwater (3 PSU) up to fully marine conditions (35 PSU) and is regarded as one of few truly brackish-water species. Na⁺/K⁺ ATPase (NAK) has been shown to be important for osmoregulation when marine organisms are challenged by changing salinities, and we therefore cloned and examined the expression of different NAKs from B. improvisus. We found two main gene variants, NAK1 and NAK2, which were approximately 70% identical at the protein level. The NAK1 mRNA existed in a long and short variant with the encoded proteins differing only by 27 N-terminal amino acids. This N-terminal stretch was coded for by a separate exon, and the two variants of NAK1 mRNAs appeared to be created by alternative splicing. We furthermore showed that the two NAK1 isoforms were differentially expressed in different life stages and in various tissues of adult barnacle, i.e the long isoform was predominant in cyprids and in adult cirri. In barnacle cyprid larvae that were exposed to a combination of different salinities and pCO₂ levels, the expression of the long NAK1 mRNA increased relative to the short in low salinities. We suggest that the alternatively spliced long variant of the Nak1 protein might be of importance for osmoregulation in B. improvisus in low salinity conditions.     

Effects of low salinity media on growth, condition, and gill ion transporter expression in juvenile Gulf killifish, Fundulus grandis

The Gulf killifish, Fundulus grandis, is a euryhaline teleost which has important ecological roles in the brackish-water marshes of its native range as well as commercial value as live bait for saltwater anglers. Effects of osmoregulation on growth, survival, and body condition at 0.5, 5.0, 8.0 and 12.0‰ salinity were studied in F. grandis juveniles during a 12-week trial. Relative expression of genes encoding the ion transport proteins Na⁺/K⁺-ATPase (NKA), Na⁺/K⁺/2Cl⁻ cotransporter(NKCC1), and cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel was analyzed. At 0.5‰, F. grandis showed depressed growth, body condition, and survival relative to higher salinities. NKA relative expression was elevated at 7 days post-transfer but decreased at later time points in fish held at 0.5‰ while other salinities produced no such increase. NKCC1, the isoform associated with expulsion of ions in saltwater, was downregulated from week 1 to week 3 at 0.5‰ while CFTR relative expression produced no significant results across time or salinity. Our results suggest that Gulf killifish have physiological difficulties with osmoregulation at a salinity of 0.5‰ and that this leads to reduced growth performance and survival while salinities in the 5.0-12.0‰ are adequate for normal function.     

Effect of eyestalk ablation on oxygen consumption of callinectes similis exposed to salinity changes

• 1.1. The effect of eyestalk ablation on preadults of Callinectes similis exposed to a constant salinity (30%.) and to simulated tidal changes in salinity (30-11 to 30%.) were measured.
• 2.2. In constant salinity, crabs showed a persistent respiratory rhythm, with a maximum oxygen consumption during the day. Under these conditions, ablation significantly increased the respiratory rate but not the rhythm.
• 3.3. In variable salinities, the highest respiratory rates occurred in salinities of 11 and 16%. during the night. In these crabs, ablation of eyestalks and subsequent injection of eyestalk extracts did not alter the respiration rate rhythm.
• 4.4. The circadian rhythm is controlled by the periodicity of environmental changes instead of the influence of eyestalk hormones.
• 5.5. Regulation of metabolism in C. similis associated with osmoregulation involves other neurosecretory organs.

     

Ionic and osmotic regulation of the haemolymph of the dragonfly, Libellula quadrimaculata (Odonata : Libellulidae)

Larvae of the widespread dragonfly, Libellula quadrimaculata, were adapted to a series of salt solutions, and the osmotic pressure, and sodium, potassium and chloride concentrations in the haemolymph measured. The regulation of potassium is extremely efficient over the range 0–50 m-mole/l. external concentration. Above this, larvae die. Sodium and chloride are regulated to a lesser extent, the larvae being able to withstand considerable changes in the concentration of these ions in the haemolymph. However, at higher external concentrations, the haemolymph concentration of these ions is maintained below that of the external medium. The osmotic pressure is regulated in parallel with sodium concentration over most of the range tested. However, in higher salinities, the osmotic pressure of the haemolymph does not fall below that of the external medium. This is seen as a strategy to limit the amount of drinking in saline media. Overall, the osmoregulatory system of L. quadrimaculata resembles that of brackish-water insects, rather than that of the more strictly freshwater dragonflies that have been studied.     

The physiological ecology of the supratidal amphipod Talorchestia longicornis

Physiology, behavior, habitat, and morphology are used to determine the degree of adaptation to life on land for amphipod species and systemization within the four functional groups of the family talitridae. Talorchestia longicornis is a semi-terrestrial amphipod found in the supratidal zone of estuaries. The present study investigates the physiological adaptations of this species to life on land through measurements of osmoregulation and respiration. Over the salinity range of 1-40‰, T. longicornis regulated its hemolymph hyperosmotically at low salinities and hypoosmotically at high salinities. The isosmotic point was about 27‰. Analogously, hemolymph chloride levels were well regulated being hyperionic at low salinities and hypoionic at high salinities. This species is capable of respiration in both air and water. Slopes (b values) of the relationship between weight and oxygen uptake rates ranged from 0.316 to 0.590. Oxygen uptake rates were higher in air than water and at night versus day. Q₁₀ values were slightly below 2.0 for respiration in air for amphipods, irrespective of weight. These physiological adaptations, along with its behaviors, habitat, and morphology, place T. longicornis within the Group III sandhoppers of the Talitridae.     

Effects of reduced salinity on the biochemical composition (lipid, protein) of zoea 1 decapod crustacean larvae

Effects of reduced salinities on dry weight (DW) and biochemical composition (total lipid and protein contents) of zoea 1 larvae were evaluated in four decapod crustacean species differing in salinity tolerance (Cancer pagurus, Homarus gammarus, Carcinus maenas, Chasmagnathus granulata). The larvae were exposed to two different reduced salinities (15‰ and 25‰ in C. granulata, 20‰ and 25‰ in the other species) for a long (ca. 50% of the zoea 1 moulting cycle) or a short period (16 h, starting at ca. 40% of the moulting cycle), while a control group was continually maintained in seawater (32‰).In general, the increments in dry weight, lipid and protein content were lower at the reduced salinities than in the control groups. In the zoea 1 of H. gammarus (stenohaline) and C. pagurus (most probably also stenohaline), the lipid and protein contents varied greatly among treatments: larvae exposed to low salinities exhibited very low lipid and protein contents at the end of the experiments compared to the controls. In some cases, there were negative growth increments, i.e. the larvae had, after the experimental exposure, lower lipid and protein contents than at the beginning of the experiment. C. maenas (moderately euryhaline) showed a lower variation in protein and lipid content than the above species. The zoea 1 of C. granulata (fairly euryhaline) showed the lowest variability in dry weight, protein and lipid content. Since salinity tolerance (eury- v. stenohalinity) is associated with the osmoregulatory capacity, our results suggest a relationship between the capability for osmoregulation and the degree of change in the biochemical composition of larvae exposed to variable salinities.Besides larval growth of these species should be affected by natural reductions of salinity occurring in coastal areas at different time scales. These effects may be potentially important for population dynamics since they should influence the number and quality of larvae reaching metamorphosis.     

Modification of the salinity response of wheat by the genome ofElytrigia elongatum

Summary Triticum aestivum cv. Chinese Spring wheat,Elytrigia elongatum (tall wheatgrass), and theTriticum-Elytrigia amphiploid were grown in complete nutrient culture containing, in addition, 0, 40, 80 and 120 mM NaCl. The 3 genotypes responded quite differently to increasing salinity; the Na concentration of wheat shoots increased in direct proportion to the increase in salinity of the external medium whereas the Elytrigia response was interpreted as showing high affinity for Na at low external Na (40 mM) but comparative exclusion of Na at high salinities (120 mM). In contrast, Na levels of the amphiploid were less than those of either wheat or Elytrigia under both low and high salinities. Thus the amphiploid behaved like wheat at 40 mM NaCl but more like Elytrigia at 120 mM NaCl because Na transport to the amphiploid shoot was restricted over the whole salinity range. The K concentration of the amphiploid shoot at high salinities was significantly greater than the K concentrations of either wheat or Elytrigia.     

Physiological Aspects of Adaptation of the Marine Microalga Tetraselmis (Platymonas) viridis to Various Medium Salinity

We studied the capability of the marine microalga Tetraselmis (Platymonas) viridis to adapt to low and high medium salinity. The normal NaCl concentration for growth of this alga is 0.5 M. It was shown that T. viridis cells could actively grow and maintain osmoregulation and cytoplasmic ion homeostasis in the wide range of external salt concentrations, from 0.01 to 1.2 M NaCl. Using the plasma membrane vesicles isolated from T. viridis cells grown at various NaCl concentrations (0.01, 0.05, 0.5, 0.9, and 1.2 M), we studied the formation of the phosphorylated intermediate of Na⁺-ATPase, the enzyme responsible for Na⁺ export from the cells with a mol wt of ca. 100 kD. Na⁺-ATPase was shown to function in the plasma membrane even in the cells growing at an extremely low NaCl concentration (0.01 M). When alga was grown in high-salt media, the synthesis of several proteins with molecular weights close to 100 kD was induced. The data obtained argue for the hypothesis, which was put forward earlier, that a novel Na⁺-ATPase isoform is induced by T. viridis growing at high NaCl concentrations.     

Glycerol fomation inDunaliella cells under non-growing conditions with hypertonic lithium or magnesium media

Glycerol formation ofDunaliella cells in non-growing media was investigated.Dunaliella tertiolecta andD. bioculata grew well in a NaCl medium but not at all in a LiCl or a MgCl₂ medium. When the cells originally suspended in a medium containing 0.5 M NaCl were transferred to media which contained one of 1 M NaCl, 1 M LiCl or 0.7 M MgCl₂, the intracellular glycerol content increased.D. tertiolecta cultured in either a 1 M LiCl or a 0.7 M MgCl₂ medium did not multiply, but maintained abilities to evolve O₂ in the light and absorb O₂ in thedark even after about a 5 day culture. From these results, it can be concluded that the halotolerance ofDunaliella to different kinds of salts is not directly related to osmoregulation by the glycerol formation.